Secondary channel of the RNA polymerase, a target for transcriptional regulation in bacteria

Abstract

Gene expression begins by an enzymatic complex known as RNA polymerase (RNApol). The basic unit (core) of RNApol in bacteria is formed by 5 protein subunits (α2ββ’ω). The three-dimensional structure of the RNApol defines two spaces that play a relevant role during transcription, defined as primary and secondary channel. The holoenzyme needs the binding of a σ subunit to recognise promoter sequences and initiate the transcription process.

Transcription is a dynamic process controlled at different steps. Genetic regulation during transcription initiation has been highly studied, and several mechanisms of regulation exist. However, the aim of this project is to study some aspects of the regulation during transcription elongation. It has been described that the alamone ppGpp, as well as several proteins, such as GreA, GreB or DksA, enter within the secondary channel and interact directly with the catalytic centre of the RNApol. The swap between the different factors that bind to the secondary channel of the RNApol may cause changes in the expression pattern.

It has been postulated that DksA and ppGpp act as cofactors, however, a previous study performed in our research group, indicated that the phenotype of ppGpp and DksA deficiencies were not always identical, letting us suggest that the occupancy degree of the secondary channel of the RNApol may have significant impact in the expression pattern in E. coli. The data obtained clearly indicate that upregulation of some genes, such as fliC, that occurs in absence of DksA, was the result of the vacancy of the secondary channel generated in a dksA strain rather than being the result of DksA having a direct repressor effect. We suggested that in the absence of DksA, the interactions of other proteins, such as GreA, are promoted and responsible of the upregulation observed.

In this project, functional, structural and phylogenetical studies of the protein GreA were performed to determine which amino acids are important for i) the functionality of GreA, ii) the ability to bind to the secondary channel of the RNApol or iii) the capacity to compete with other factors, such as DksA. We have determined that greA overexpression produces a negative effect of the bacterial growth. Moreover, this negative effect is enhanced in absence of DksA, highlighting the hypothesis of a competition between factors that bind into the secondary channel.

The effect of this competition between GreA and DksA was also determined studying the expression of the fliC gene. Our data showed that both, GreA and DksAare required for fliC expression but act at different levels in the regulatory cascade of flagella expression regulation. GreA may control fliC expression during transcription elongation whereas DksA may act during transcription initiation.

Changes in the amount of GreA, could affect the competition between factors that bind to the secondary channel of the RNApol. Therefore, we have determined the expression pattern of greA. Transcriptional studies showed a crosstalk between the different factors that bind into the secondary channel of the RNApol exists.

Finally, transcriptomic studies were performed to determine the effect of ppGpp and DksA on the expression pattern of Salmonella enterica serovar Typhimurium. The results obtained indicate : i) the effect of the possible competence between the factors that interact into the secondary channel of the RNApol and ii) the effect of ppGpp and DksA on the expression of several virulence factors as well as different mobile elements present in Salmonella.

El control de l’expressió gènica en bacteris recau principalment sobre un complex enzimàtic anomenat ARN polimerasa (ARNpol). A procariotes, la seva unitat bàsica (core) està formada per 5 subunitats proteiques (a2bb’w). S’han determinat dos canals entre les diferents subunitats de l’ARNpol: el canal primari, on es desenvolupa la transcripció, i el canal secundari, que comunica el medi exterior amb el centre catalític de l’ARNpol. Tot i així, aquest holoenzim necessita la unió d’una subunitat σ per ser capaç de reconèixer una seqüència promotora i iniciar la transcripció. S’han descrit diferents factors, tant proteics com no proteics, que poden interaccionar amb el canal secundari de l’ARNpol i causar alteracions a l’expressió gènica. En aquesta tesi ens hem centrat en la possible competència entre els diferents factors que poden interaccionar amb el canal secundari de l’ARNpol.

Estudis anterior duts a terme en el nostre grup d’investigació, ens van permetre postular una possible competència entre els diferents factors que interaccionen amb el canal secundari de l’ARNpol, més concretament entre les proteïnes GreA i DksA. Aquesta competència provocaria alteracions en el patró d’expressió gènica d’Escherichia coli.

En aquest treball s’han dut a terme estudis funcionals, estructurals i filogenètics de la proteïna GreA que ens han permès determinar quins aminoàcids, i com a conseqüència quins dominis, podrien ser importants per la funcionalitat de la proteïna, la seva capacitat d’unir-se a l’ARNpol i la seva capacitat de competir amb altres factors. A més, hem estudiat quin efecte té la competència entre els diferents factors que interaccionen amb el canal secundari sobre l’expressió d’un gen diana.

Canvis en els nivells de la proteïna GreA, poden afectar la competència pel canal secundari de l’ARNpol Per això hem determinat el patró d’expressió del gen greA, així com l’existència d’una regulació creuada entre les diferents proteïnes que interaccionen amb el canal secundari.

Finalment, hem dut a terme un estudi transcriptòmic en Salmonella enterica serovar Typhimurium, amb l’objectiu de determinar quin és l’efecte d’aquesta competència en l’expressió de factors de virulència.